Reversible platelet inhibition

ABSTRACT

The present invention relates, in general, to receptors and to platelet aggregation and, in particular, to a method of inhibiting platelet aggregation using an aptamer that binds to and inhibits the activity of a receptor, such as glycoprotein IIb/IIIa (gpIIb/IIIa), and to aptamers suitable for use in such a method. The invention also relates to antidotes to antiplatelet agents and to methods of using such antidotes to reverse aptamer-induced platelet inhibition. The invention further relates to von Willebrand Factor (VWF) inhibitors, and antidotes therefore, and to methods of using same.

This is a continuation-in-part of U.S. application Ser. No. 12/311,943,filed Mar. 31, 2010, which is the U.S. national phase of InternationalApplication No. PCT/US2007/022358, filed Oct. 19, 2007, which designatedthe U.S. and claims priority from Provisional Application No.60/852,650, filed Oct. 19, 2006, the entire contents of whichapplications are incorporated herein by reference.

This invention was made with government support under Grant No. NHLB1RO1 HL65222 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

TECHNICAL FIELD

The present invention relates, in general, to receptors and to plateletaggregation and, in particular, to a method of inhibiting plateletaggregation using an aptamer that binds to and inhibits the activity ofa receptor, such as glycoprotein IIb/IIIa (gpIIb/IIIa), and to aptamerssuitable for use in such a method. The invention also relates toantidotes to antiplatelet agents and to methods of using such antidotesto reverse aptamer-induced platelet inhibition. The invention furtherrelates to von Willebrand Factor (VWF) inhibitors, and antidotestherefore, and to methods of using same.

BACKGROUND

Inhibitors of gpIIb/IIIa have proven to be efficacious asanti-thrombotic agents for use in treatment of cardiovascular disease.Abciximab, a chimeric human-murine monoclonal antibody, was the firstgpIIb/IIIa antagonist developed (Binkley et al, Nucleic Acid Research23:3198-3205 (1995)). Eptifibatide, a small peptide, and Tirofiban, asmall non-peptide, both interact with and inhibit the function of thebeta-3 (β₃) sub-unit of gpIIb/IIIa (Scarborough et al, J. Biol. Chem.268:1066-1073 (1993), Bednar et al, J. Pharmacol. Exp. Ther.285:1317-1326 (1998), Hartman et al, J. Med. Chem. 35:4640-4642 (1992)).The two main drugs used clinically are Abciximab and Eptifibatide.

Abciximab is approved for use in patients undergoing percutaneouscoronary intervention (PCI) and is being studied for use in acutecoronary syndromes (ACS). The EPIC trial revealed that Abciximab reducedthe morbidity and mortality of cardiovascular disease, but also showedan increase in major bleeding episodes from 7% to 14% and an increase inblood transfusions from 10% to 21% (Lincoff et al, Am. J. Cardiol.79:286-291 (1997)). Eptifibitide is also used in PCI and, likeAbciximab, is an effective antithrombotic with a trend towards increasedbleeding (The PURSUIT Trial Investigators, N. Eng. J. Med. 339:436-443(1998)). In addition to bleeding complications, readministration is apotential concern, especially with Abciximab, where initialadministration was associated with a human antichimeric antibodyresponse in 7% of patients (Tcheng, Am. Heart J. 139:S38-45 (2000)).Finally, thrombocytopenia is also seen in patients who receivegpIIb/IIIa antagonists. Severe thrombocytopenia (<20,000/μl) occurs inalmost 0.5% of patients after intravenous administration (Topol et al,Lancet 353:227-231 (1999)). The most pressing issue with these drugs,given the clinical environment in which they are used, is the need toturn off or reverse their activity quickly. This would allow physiciansto reduce the side effects of the medications should they become a riskto the health and safety of the patient and would also allow surgeons toperform immediate coronary bypass graft surgery, should the need arise.Thus, the development of new gpIIb/IIIa inhibitors with matchedantidotes is a medical priority.

Ribonucleic acid ligands, or aptamers, are a new class of drug compoundsideally suited to anticoagulation therapy. They bind to their targetswith high affinity and specificity, are only slightly immunogenic andtheir bioavailability can be tailored to suit a particular clinical need(Nimjee et al, Annu. Rev. Med. 56:555-583 (2005)). More recently,research has shown that these drugs can be controlled with antidotesboth in vitro and in vivo (Nimjee et al, Molecular Therapy: the Journalof the American Society of Gene Therapy (2006), Mol. Ther. 14:408-45Epub Jun. 9, 2006, Rusconi et al, Nat. Biotechnol. 22:1423-1428 (2004),Rusconi et al, Nature 419:90-94 (2002)).

The present invention relates to RNA ligands (aptamers) that inhibitreceptor function and activity, including platelet function andactivity. The invention further relates to specific, rationally-designedantidotes that can reverse this inhibitory effect.

SUMMARY OF THE INVENTION

In general, the present invention relates to inhibitors of plateletaggregation. More specifically, the invention relates to RNA ligands oraptamers that can inhibit the activity of a receptor, such asgpIIb/IIIa, as well as aptamers that inhibit VWF, and to methods ofusing same. The invention additionally relates to agents (antidotes)that can reverse the inhibitory effect of such ligands/aptamers.

Objects and advantages of the present invention will be clear from thedescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Affinity of rounds to platelets. While the progress of theselection to gpIIb/IIIa was monitored by real-time PCR, the binding wasmeasured on whole platelets using 32P RNA and nitrocellulosepartitioning scheme. The [gpIIb/IIIa] was determined by assuming 80,000gpIIb/IIIa molecules per platelet (Tcheng, Am. Heart J. 139:S38-45(2000)). ▪=Sel2 library; ▴=round 4; ▾=round 8; ♦=round 12.

FIG. 2: Round 12 clones binding to platelets. Clones from round 12 boundto gpIIb/IIIa on platelets with different affinity. C1=▪; C2=▴; C3=▾;C4=♦; C5=●; C6=▪; C7=▴; C8=▾; C9=♦; C10=●.

FIGS. 3A-3C: Functional activity of aptamers. Aptamers were tested in aPFA-100. All clones were tested in a volume of 840 μl at a finalconcentration of 1 μM (FIG. 3A). The ability of the aptamers to inhibitplatelet function in pig blood was evaluated (FIG. 3B). Plateletactivity of Cl was tested in a Chronolog Lumi-aggregometer (FIG. 3C).Error bars represent S.E.M.

FIG. 4: Aptamer competes with current drugs for binding to gpIIb/IIIa.The assay was carried out in 3-fold serial dilutions between 100 to0.1-fold excess of each compound's dissociation constant. ▪=Abciximab;▴=Eptifibatide; ▾=Aptamer.

FIGS. 5A and 5B: Antidote reverses aptamer activity in PFA. FIG. 5A.Antidote oligonucleotides (SEQ ID NOs:40-44, respectively, in order ofappearance) were designed to portions of the variable region of CI-6(SEQ ID NO:39). FIG. 5B. Modified (2′-Omethyl) antidote oligonucleotides(AO) designed against distinct regions of the aptamer. AO2 representedthe most effective inhibitor with a closing time of 81±19.5 s, while AO5was the least effective, with a closing time of 129.5±14.5 s; error barsrepresent S.E.M.

FIG. 6. Binding improved over consecutive rounds of VWF selection.Nitrocellulose filter binding assay with ³²P labeled RNA molecules.Inverted triangles (▾) represent the original RNA library (Sel2).Squares (▪) represent round 5, triangles (▴) represent round 7 anddiamonds (⋄) represent round 9 RNA pools. Y-axis is the fraction of RNAmolecules bound at a given VWF protein concentration. Proteinconcentration given in micro molar (X-axis).

FIG. 7. Clone VWF R9.14 inhibits platelet activity in a PFA-100: VWFaptamer R9.14 was added to 800 microL whole blood at increasing isconcentrations and a PFA-100 assay was performed to determine if theaptamer delayed platelet mediated closing. Integrilin is positivecontrol. Each point has been performed in duplicate. Error barsrepresent the range of data.

FIG. 8. Antidote Sel 2 3′W1 reverses VWF R9.14 activity in a PFA-100:VWF aptamer R9.14 was added to 800 microL whole blood at 40 nMconcentration, incubated for 5 minutes. Than, the antidote added at 50×molar excess. After an additional 5 minute incubation, a PFA-100 assaywas performed to determine if the antidote reversed the VWF R9.14aptamer activity. @95C columns are positive control. VWF R9.14 T7 is amutant aptamer used as negative control. Each point has been performedin duplicate. Error bars represent the range of data.

FIGS. 9A-9D. “Convergent” SELEX yielded aptamers that bind to VWF withhigh affinity. FIG. 9A) Progress of the “convergent” SELEX was followedusing a nitrocellulose filter binding assay. Inverted triangles (▾)represent the starting RNA library (Sel2). Squares (▪) represent theplasma focused library. Triangles (▴) represent “convergent” SELEX round2 and diamonds (♦) represent “convergent” SELEX round 4. The X-axisrepresents VWF concentration and the Y-axis represents the fraction ofRNA bound to the protein. FIG. 9B) Binding affinities of VWF aptamersR9.3, R9.4 and R9.14 were determined using a nitrocellulose filterbinding assay. Squares (▪) represent R9.3, triangles (▴) represent R9.4and inverted triangles (▾) represent R9.14. Each data point was done intriplicate; error bars represent the SEM (standard error of the mean) ofthe data. FIG. 9C) Binding of aptamers to VWF, VWF SPI and VWF SPIIIfragments was determined using a nitrocellulose filter binding assay.Aptamers R9.3 and R9.14 bind to both full length VWF and the VWF SPIIIfragment but not to the VWF SPI fragment. Aptamer R9.4 binds to fulllength VWF, the VWF SPIII and the VWF SPI fragment. FIG. 9D) Cartoondepicting the VWF, its subunits and SP I and SP III fragments.

FIGS. 10A-10C. VWF aptamers R9.3 and R9.14 inhibit platelet aggregationby blocking the VWF-GP Ib-IX-V interaction. FIG. 10A) The function ofVWF aptamers R9.3, R9.4 and R9.14 was measured at a 1 μM concentrationin a PFA-100 assay. Platelet buffer and starting aptamer library (Sel2)were used as negative controls. Error bars represent the range of data.Each data point was done in triplicate. FIG. 10B) Varying concentrationsof VWF aptamers R9.3 and R9.14 were added to normal whole blood; closingtimes were measured in a PFA-100 assay using collagen/ADP cartridges.Error bars represent the range of data. Each data point was done intriplicate. FIG. 10C) VWF aptamers R9.3 and VWF R9.14 were tested inristocetin, collagen, ADP and thrombin (SFLLRN) induced plateletaggregation. Filled bars represent percent aggregation in normalplatelet rich plasma. Error bars represent the range of data; each datapoint was done in triplicate.

FIGS. 11A and 11B. Antidote oligonucleotides to R9.14 can inhibitaptamer binding to VWF. FIG. 11A) Cartoon depicting the antidote designto aptamer VWF R9.14 (SEQ ID NO:45). Black bars depict the positions ofsequence complementarities. FIG. 11B) Reversal of aptamer VWF R9.14binding to VWF was accomplished by antidote oligonucleotide 6 (AO6)(triangles) but not by AO5 (inverted triangles). AO6 and AO5 together(diamonds) also inhibit aptamer binding to VWF. The starting library(Sel2; circles) was used as a control.

FIG. 12A-12C. Antidote oligonucleotides to aptamer VWF R9.14 can reverseaptamer function rapidly and for a prolonged period of time. FIG. 12A)AO6 completely reverses aptamer function in a PFA-100 assay (black bars)at a 40:1 ratio. A scrambled antidote oligonucleotide is used as anegative control (grey bars). Error bars represent the range of data.Each data point was done in triplicate. FIG. 12B) AO6 achieved completereversal of aptamer VWF R9.14 function in a PFA-100 assay in 2 minutes.AO6 was used at 40:1 ratio to VWF R9.14 (40 nM). Error bars representthe range of data. Each data point was done in triplicate. FIG. 12C) AO6inhibits aptamer VWF R9.14 function for 4 hours in a PFA-100 assay(black bars). A scrambled antidote oligonucleotide was used as anegative control (grey bars). Error bars represent the range of data.Each data point was done in triplicate.

FIGS. 13A and 13B: FIG. 13A: VWF Aptamer truncate 9.14-T10 binds withsimilar affinity compared to full length aptamer. Binding was performedusing a nitrocellulose-filter binding assay. Circles (●) representfull-length aptamer VWF. 9.14. Squares (▪) represent aptamer VWF9.14-T10. The X-axis represents VWF concentration and the Y-axisrepresents the fraction of RNA bound to the protein. FIG. 13B: VWFAptamer 9.14 and derivatives inhibit platelet activity in a PFA-100assay. The inhibitory activities of VWF aptamers 9.14, 9.14-T10 andCh-9.14-T10 were measured at a 50 nM concentration in a PFA-100 assay.Error bars represent the mean±SEM. Each data point was performed induplicate.

FIGS. 14A-14C: FIG. 14A: VWF Aptamer Ch-9.14-T10-treated animalsmaintained carotid artery patency following ferric chloride induceddamage of murine carotid arteries. Transit time or blood flow (ml/min)through the damaged carotid artery did not decline in animals treatedwith the aptamer compared to control animals treated with PBS whosecarotid blood flow ceased during the course of the experiment (n=11 pergroup). Squares (▪) represent aptamer Ch-9.14-T10-treated animals. Opencircles (∘) represent phosphate-buffered saline (PBS)-treated animals.No significant change was observed in the blood flow in theaptamer-treated group during the course of the experiment. However, asignificant difference in blood flow between the aptamerCh-9.14-T10-treated mice and mice that did not receive the aptamer wasobserved (p<0.0001). X-axis represents time in minutes (min) over whichthe experiment took place. Y-axis represents transit time or blood flowin milliliters per minute (ml/min). A transit time of 0 indicates thatthe carotid artery is completely occluded. Error bars representmean±SEM. FIGS. 14B and 14C: Carotid arteries of AptamerCh-9,14-T10-treated (FIG. 14B) animals maintained 100% patency while allanimals that did not receive the aptamer (FIG. 14C) had thrombi thatcompletely occluded their damaged carotid arteries (p<0.0001) (n=11 pergroup). Histopathological sections were stained with hematoxylin andeosin.

FIG. 15: VWF Aptamer Ch-9.14-T10 inhibits platelet plug formation inmice (n=5). Mice (18-24 g) were treated with 10, 3, 1 and 0.3 mg/kg ofaptamer, then the distal 2 mm of their tails were clipped and blood lossmeasured. Animals treated with all doses of aptamer had significantblood loss when surgically challenged compared to animals not given theaptamer (p<0.0001). Y-axis represented blood loss in microliters (μl).Error bars represent the mean±SEM.

FIGS. 16A-16D: Antidotes for VWF aptamer Ch-9.14-T10. FIG. 16A: Antidoteoligonucleotide (SEQ ID NOs:68-74, respectively, in order of appearance)design based upon Watson-Crick base-pairing to aptamer VWF Ch-9.14-T10(SEQ ID NO:67) (top). FIG. 16B: Antidote oligonucleotide 1 (AO1)completely reverses the activity of Aptamer Ch-9.14-T10 (50 nM) asmeasured in the PFA-100 assay. Each antidote oligonucleotide (AO) wasadded in 50-fold molar excess over aptamer Ch-9.14-T10. Y-axis representclosing time in seconds (s). Error bars represent mean and SEM. Allmeasurements were done in duplicate. FIG. 16C: AO1 and FIG. 16D: theUniversal antidote CDP reverse that activity of Aptamer Ch-9.14-T10 in aPFA-100 assay at 10-fold molar excess over aptamer. AO1 and CDP wereadded in a 5 to 20-fold molar excess of 50 nM aptamer Ch-9.14-T10.Negative control samples that did not receive an antidote were treatedwith phosphate-buffered saline. Y-axis represent closing time in seconds(s). Error bars represent mean and SEM. All measurements were done induplicate.

FIGS. 17A and 17B: Antidote control of VWF aptamer activity in vivo.FIG. 17A: AO1 or CDP can reverse the activity of aptamer Ch-9.14-T10 insurgically challenged animals. Mice (n=5 per group) were treated withaptamer Ch-9.14-T10 (3 mg/kg) by intraperitoneal injection. AO1 or CDPwere administered at a10-fold molar excess over aptamer via tail veininjection, then the animals were surgically challenged by clipping theirtails and blood loss was measured. Animals that did not receive anantidote experienced significant blood loss compared to those giveneither or the antidotes (AO1- and CDP compared to no antidote,p<0.0001). However, no significant difference was observed in blood lossfrom animals given the aptamer and either of the antidotes (AO1-treatedor CDP-treated) and animals not given the aptamer (p=0.74). FIG. 17B:CDP reverses the activity of the anti-FIXa aptamer Ch9.3T aptamer andthe VWF aptamer Ch-9.14-T10 simultaneously. Mice (n=5 per group) weretreated with the VWF aptamer (3 mg/kg) alone, the anti-FIXa aptamer (10mg/kg) alone or both aptamers. Animals were then given antidoteoligonucleotides A01 (for VWF aptamer) or 5-2C (for FIXa aptamer), theuniversal antidote CDP or no antidote (PBS vehicle control) andsurgically challenged. Administration of antidote oligonucleotides A01and 5-2C to animals that received both aptamers decreased blood losssignificantly compared to the negative control antidote (p=0.03) butthis was well above blood loss levels from animals that did not receiveeither aptamer. Blood loss from animals that received both aptamers andthe universal antidote (CDP) was similar to that from mice that did notreceive either aptamer (untreated, p=0.27) (Y-axis shows blood loss inmicroliters (μl)). Error bars represent the mean±SEM.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to aptamers (DNA or RNA) thatcan bind to receptors and inhibit cell-cell or cell-particleinteractions. The present invention relates, more specifically, toantiplatelet compounds (e.g., aptamers (DNA or RNA) and to methods ofusing same in the treatment of, for example, cardiovascular disease. Ina preferred embodiment, the invention relates to RNA ligands or aptamersthat can: i) bind to and inhibit the activity of gpIIb/IIIa, an integrinon the surface of platelets that is principally responsible for plateletaggregation, or ii) bind to VWF, a multimeric blood glycoproteininvolved in coagulation, and inhibit platelet adhesion and aggregation.The invention also relates to antidote molecules that can bind to andreverse aptamer-induced platelet inhibition. The antiplateletagent/antidote pairs of the present invention provide physicians withenhanced control over antithrombotic therapy.

Aptamers suitable for use as antiplatelet compounds (e.g., via theirability to bind to and inhibit the activity of gpIIb/IIIa or theirability to bind to VWF) and be prepared using SELEX methodology (see,for example, U.S. Pat. Nos. 5,270,163, 5,817,785, 5,595,887, 5,496,938,5,475,096, 5,861,254, 5,958,691, 5,962,219, 6,013,443, 6,030,776,6,083,696, 6,110,900, 6,127,119, 6,147,204, U.S. Appln 20030175703 and20030083294, Potti et al, Expert Opin. Biol. Ther. 4:1641-1647 (2004),Nimjee et al, Annu. Rev. Med. 56:555-83 (2005)). The SELEX processconsists of iterative rounds of affinity purification and amplificationof oligonucleotides from combinatorial libraries to yield high affinityand high specificity ligands. Combinatorial libraries employed in SELEXcan be front-loaded with 2′ modified RNA nucleotides (e.g., 2′fluoro-pyrimidines) such that the aptamers generated are highlyresistant to nuclease-mediated degradation and amenable to immediateactivity screening in cell culture or bodily fluids.

Specific aptamers suitable for use as antiplatelets are described in theExamples that follow.

Aptamers of the invention can be used in the treatment of acardiovascular disease in humans and non-human animals. For example,these aptamers can be used in patients undergoing PCI and can be used inthe treatment of ACS (including stroke and arterial thrombosis). Use ofthe instant aptamers is expected to significantly reduce the morbidityand mortality associated with thrombosis.

The present invention also relates to antidotes for the antiplateletaptamers described herein. These antidotes can comprise oligonucleotidesthat are reverse complements of segments of the antiplatelet aptamers.In accordance with the invention, the antidote is contacted with thetargeted aptamer under conditions such that it binds to the aptamer andmodifies the interaction between the aptamer and its target molecule(e.g., gpIIb/IIIa or VWF). Modification of that interaction can resultfrom modification of the aptamer structure as a result of binding by theantidote. The antidote can bind free aptamer and/or aptamer bound to itstarget molecule.

Antidotes of the invention can be designed so as to bind any particularaptamer with a high degree of specificity and a desired degree ofaffinity. The antidote can be designed so that upon binding to thetargeted aptamer, the three-dimensional structure of that aptamer isaltered such that the aptamer can no longer bind to its target moleculeor binds to its target molecule with less affinity.

Antidotes of the invention include any pharmaceutically acceptable agentthat can bind an aptamer and modify the interaction between that aptamerand its target molecule (e.g., by modifying the structure of theaptamer) in a desired manner. Examples of such antidotes includeoligonucleotides complementary to at least a portion of the aptamersequence (including ribozymes or DNAzymes or peptide nucleic acids(PNAs)), nucleic acid binding peptides, polypeptides or proteins(including nucleic acid binding tripeptides (see, generally, Hwang etal, Proc. Natl. Acad. Sci. USA 96:12997 (1999)), and oligosaccharides(e.g., aminoglycosides (see, generally, Davies et al, Chapter 8, p. 185,RNA World, Cold Spring Harbor Laboratory Press, eds Gestlaad and Atkins(1993), Werstuck et al, Science 282:296 (1998), U.S. Pat. Nos. 5,935,776and 5,534,408). (See also Chase et al, Ann. Rev. Biochem. 56:103 (1986),Eichhorn et al, J. Am. Chem. Soc. 90:7323 (1968), Dale et al,Biochemistry 14:2447 (1975) and Lippard et al, Acc. Chem. Res. 11:211(1978)).

Standard binding assays can be used to screen for antidotes of theinvention (e.g., using BIACORE assays). That is, candidate antidotes canbe contacted with the aptamer to be targeted under conditions favoringbinding and a determination made as to whether the candidate antidote infact binds the aptamer. Candidate antidotes that are found to bind theaptamer can then be analyzed in an appropriate bioassay (which will varydepending on the aptamer and its target molecule) to determine if thecandidate antidote can affect the binding of the aptamer to its targetmolecule.

In a preferred embodiment, the antidote of the invention is anoligonucleotide that comprises a sequence complementary to at least aportion of the targeted aptamer sequence. Advantageously, the antidoteoligonucleotide comprises a sequence complementary to 6-25 consecutivenucleotides of the targeted aptamer, preferably, 8-20 consecutivenucleotides, more preferably, 10-15 consecutive nucleotides.

Formation of duplexes by binding of complementary pairs of shortoligonucleotides is a fairly rapid reaction with second orderassociation rate constants generally between 1×10⁶ and 3×10⁶ M⁻¹ s⁻¹.Thus, the effect on an aptamer by formation of a duplex with acomplimentary oligonucleotide is rapid. Stability of short duplexes ishighly dependent on the length and base-composition of the duplex. Thethermodynamic parameters for formation of short nucleic acid duplexeshave been rigorously measured, resulting in nearest-neighbor rules forall possible base pairs such that accurate predictions of the freeenergy, T_(m) and thus half-life of a given oligoribonucleotide duplexcan be calculated (e.g., Xia et al, Biochem. 37:14719 (1998) (see alsoEguchi et al, Antisense. RNA, Annu. Rev. Biochem. 60:631 (1991)).

Antidote oligonucleotides of the invention can comprise modifiednucleotides that confer improved characteristics, such as improved invivo stability and/or improved delivery characteristics. Examples ofsuch modifications include chemical substitutions at the sugar and/orbackbone and/or base positions. Oligonucleotide antidotes can containnucleotide derivatives modified at the 5- and 2′ positions ofpyrimidines, for example, nucleotides can be modified with 2′ amino,2′-fluoro and/or 2′-O-methyl. Modifications of the antidoteoligonucleotides of the invention can include those that provide otherchemical groups that incorporate additional charge, polarization,hydrophobicity, hydrogen bonding and/or electrostatic interaction. Suchmodifications include but are not limited to, 2′ position sugarmodifications, locked nucleic acids, 5 position pyrimidinemodifications, 8 position purine modifications, modification atexocyclic amines, substitution of 4-thiouridine, substitution of 5-bromoor 5-iodo-uracil, backbone modifications, phosphorothioate or alkylphosphate modifications, methylations, unusual base-pairing combinationssuch as isobases isocytidine and isoguanidine, etc. Modifications canalso include 3′ and 5′ modifications, such as capping, and addition ofPEG or cholesterol. (See also Manoharan, Biochem. Biophys. Acta 1489:117(1999); Herdewijn, Antisense Nucleic Acid Drug Development 10:297(2000); Maier et al, Organic Letters 2:1819 (2000)).

A typical aptamer possesses some amount of secondary structure—itsactive tertiary structure is dependent on formation of the appropriatestable secondary structure. Therefore, while the mechanism of formationof a duplex between a complementary oligonucleotide antidote of theinvention and an aptamer is the same as between two short linearoligoribonucleotides, both the rules for designing such interactions andthe kinetics of formation of such a product are impacted by theintramolecular aptamer structure. The rate of nucleation is importantfor formation of the final stable duplex, and the rate of this step isgreatly enhanced by targeting the oligonucleotide antidote tosingle-stranded loops and/or single-stranded 3′ or 5′ tails present inthe aptamer. For the formation of the intermolecular duplex to occur,the free energy of formation of the intermolecular duplex has to befavorable with respect to formation of the existing intramolecularduplexes within the targeted aptamer. Thus, oligonucleotide antidotes ofthe invention are advantageously targeted at single-stranded regions ofthe aptamer. This facilitates nucleation and, therefore, the rate ofaptamer activity modulation, and also, generally leads to intermolecularduplexes that contain more base pairs than the targeted aptamer.

Various strategies can be used to determine the optimal site foroligonucleotide binding to a targeted aptamer. An empirical strategy canbe used in which complimentary oligonucleotides are “walked” around theaptamer. In accordance with this approach, 2′Omethyl oligonucleotides(e.g., 2′Omethyl oligonucleotides) about 15 nucleotides in length can beused that are staggered by about 5 nucleotides on the aptamer (e.g.,oligonucleotides complementary to nucleotides 1-15, 6-20, 11-25 etc. ofaptamer 9.3t). An empirical strategy may be particularly effectivebecause the impact of the tertiary structure of the aptamer on theefficiency of hybridization can be difficult to predict. Assaysdescribed, for example, iii U.S. Appln. No. 20030083294 can be used toassess the ability of the different oligonucleotides to hybridize to aspecific aptamer, with particular emphasis on the molar excess of theoligonucleotide required to achieve complete binding of the aptamer. Theability of the different oligonucleotide antidotes to increase the rateof dissociation of the aptamer from its target molecule can also bedetermined by conducting standard kinetic studies using, for example,BIACORE assays. Oligonucleotide antidotes can be selected such that a5-50 fold molar excess of oligonucleotide, or less, is required tomodify the interaction between the aptamer and its target molecule inthe desired manner.

Alternatively, the targeted aptamer can be modified so as to include asingle-stranded tail (3′ or 5′) in order to promote association with anoligonucleotide modulator. Suitable tails can comprise 1 to 20nucleotides, preferably, 1-10 nucleotides, more preferably, 1-5nucleotides and, most preferably, 3-5 nucleotides (e.g., modifiednucleotides such as 2′Omethyl sequences). Tailed aptamers can be testedin binding and bioassays (e.g., as described in U.S. Appln. No.20030083294) to verify that addition of the single-stranded tail doesnot disrupt the active structure of the aptamer. A series ofoligonucleotides (for example, 2′Omethyl oligonucleotides) that canform, for example, 1, 3 or 5 basepairs with the tail sequence can bedesigned and tested for their ability to associate with the tailedaptamer alone, as well as their ability to increase the rate ofdissociation of the aptamer from its target molecule.

The present invention relates to antidotes that specifically and rapidlyreverse the anticoagulant and antithrombotic effects of aptamers thattarget gpIIb/IIIa and VWF. In accordance with this embodiment, antidotes(advantageously, oligonucleotide inhibitors) are administered thatreverse the aptamer activity.

At least three clinical scenarios exist in which the ability to rapidlyreverse the activity of an antithrombotic, anticoagulant or antiplateletaptamer is desirable. The first case is when anticoagulant orantithrombotic treatment leads to hemorrhage. The potential formorbidity or mortality from this type of bleeding event can be asignificant risk. The second case is when emergency surgery is requiredfor patients who have received antithrombotic treatment. This clinicalsituation can arise, for example, in patients who require emergencycoronary artery bypass grafts while undergoing PCI under the coverage ofgpIIb/IIIa inhibitors. The third case is when an anticoagulant aptameris used during a cardiopulmonary bypass procedure. Bypass patients arepredisposed to post operative bleeding. In each case, acute reversal ofthe anticoagulant effects of an aptamer via an antidote (e.g., anoligonucleotide antidote targeted to an anticoagulant or antithromboticaptamer) allows for improved, and likely safer, medical control of theanticoagulant or antithrombotic compound.

The aptamers and antidotes of the invention can be formulated intopharmaceutical compositions that can include, in addition to the aptameror antidote, a pharmaceutically acceptable carrier, diluent orexcipient. The precise nature of the composition will depend, at leastin part, on the nature of the aptamer or antidote and the route ofadministration. Optimum dosing regimens can be readily established byone skilled in the art and can vary with the aptamer and antidote, thepatient and the effect sought. Because the antidote activity is durable,once the desired level of modulation of the aptamer by the antidote isachieved, infusion of the antidote can be terminated, allowing residualantidote to clear the human or non-human animal. This allows forsubsequent re-treatment of the human or animal with the aptamer asneeded. Alternatively, and in view of the specificity of antidoteoligonucleotides of the invention, subsequent treatment can involve theuse of a second, different aptamer/antidote oligonucleotide pair.

The aptamers and antidotes can be administered directly (e.g., alone orin a liposomal formulation or complexed to a carrier (e.g., PEG)) (seefor example, U.S. Pat. No. 6,147,204 for examples of lipophiliccompounds and non-immunogenic high molecular weight compounds suitablefor formulation use). Alternatively, oligonucleotide antidotes of theinvention can be produced in vivo following administration of aconstruct comprising a sequence encoding the oligonucleotide. Techniquesavailable for effecting intracellular delivery of RNA antidotes of geneexpression can be used (see generally Sullenger et al, Mol. Cell. Biol.10:6512 (1990)). (Also incorporated by reference is the followingcitation that describes APTT and other clotting assays: Quinn et al, J.Clin. Lab. Sci. 13(4):229-238 (2000). This review describes theproperties and biochemistry of various clotting assays including APTT,PT and thrombin time assays, and their use in diagnosingcoagulopathies.)

In addition to antidote oligonucleotides described above, and methods ofusing same, the invention also relates to the use of antidotes that bindin a sequence independent manner described, for example, in U.S.Provisional Application No. 60/920,807 and to method of using same tomodulate (e.g., reverse or inhibit) the activity of aptamers describedherein (see also application Ser. No. 12/588,016).

Certain aspects of the invention can be described in greater detail inthe non-limiting Examples that follows. (See also Oney et al,Oligonucleotides 17:265-274 (2007)).

EXAMPLE 1

Experimental Details

Binding gpIIb/IIIa to plates

An enzyme linked immunosorbant assay (ELISA) was used to assessgpIIb/IIIa adherence to Immulon 4HBX plates. Briefly, 100 ρmolgpIIb/IIIa was incubated with the Immulon 4HBX plates at 4° C.overnight. After washing 5× with TMB buffer (20 mM Hepes, pH: 7.4; 120mM NaCl; 5 nM KCl; 1 mM CaCl₂; 1 mM MgCl₂; 0.01% BSA), wells wereblocked with 1% BSA at room temperature for 1 h. The wells were washed5× and incubated at 37° C. for 2 hrs with 10 μg/mL CD41, a mouseanti-human antibody that recognizes the gpIIb/IIIa complex), CD61 (amouse anti-human antibody that recognizes the β₃-subunit of the protein(Southern Biotechnology Associates, Birmingham, Ala.)) or buffer. Afterwashing 5×, 1:80,000 (v/v) goat anti-mouse IgG-HRP (JacksonImmunoResearch Laboratories, Inc., West Grove, Pa.) was added andincubated at room temperature for 2 h. The wells were washed 5× and TMBsubstrate (Sigma-Aldrich, St. Louis, Mo.) was added. The plate wascovered with aluminum foil and placed on a shaker for 15 min. In orderto quench the reaction, 0.1 M sulfuric acid was added and the plate wasscanned at 450 nm using an EL311 Microplate Autoreader (Bio-tekInstruments, Inc., Winooski, Vt.).

Selection of RNA Ligands to gpIIb/IIIa

Using the data from the gpIIb/IIIa bound to platelets, 100 μmol ofgpIIb/IIIa (Enzyme Research, South Bend, Ind.) in HEPES-buffered salineand 1 mM CaCl₂ was bound to Immulon 4HBX plates (Thermo ElectronCorporation, Boston, Mass.) at 4° C. overnight. The plates were thenwashed 3× with binding buffer (20 mM HEPES, pH: 7.4; 120 mM NaCl; 5 nMKCl; 1 mM CaCl₂; 1 mM MgCl₂; 0.01% BSA) and blocked with 3% BSA at roomtemperature for 1 h. In order to pre-clear plate-binding aptamers,no-protein wells (i.e., wells that had no gpIIb/IIIa in them) were alsoblocked with BSA and, after washing, RNA was added to nude wells andincubated at 37° C. for 1 h. The protein-blocked wells were washed 3×with binding buffer and the RNA from the nude wells was transferred tothe protein-coated wells and incubated at 37° C. for 2 h. The wells werewashed 3× and 75 μL of elution buffer (10 mM HEPES pH: 7.4; 120 mM NaCl;5 mM KCl; 5 mM EDTA pH: 8.0) was added to wells and incubated at 37° C.for 30 min before being transferred to tubes. The eluted RNA ligandswere reverse-transcribed and amplified as described (Drolet et al,Combinatorial Chemistry & High Throughput Screening 2:271-278 (1999)).

Binding Assays

Aptamer binding to purified platelets. Platelets were purified fromfreshly drawn blood from healthy volunteers (Hoffman et al, Am. J. Clin.Pathol. 98:531-533 (1992)). Briefly, platelets were isolated by densitygradient centrifugation, then separated from plasma proteins bygel-filtration over 50 mL column of Sepharose Cl-2B in Tyrodes buffer(15 mM HEPES, pH 7.4; 3.3 mM Na₂PO₄; 138 mM NaCl, 2.7 mM KCl; 1 mMMgCl₂, 5.5 mM dextrose) with 1 mg/mL bovine serum albumin. The plateletswere activated prior to binding with 1 nM thrombin; 1 mM CaCl₂ and 200ng/mL convulxin.

Dissociation constants (K_(d)) were determined using a double-filter,nitrocellulose binding method (Wong et al, Proc. Natl. Acad. Sci. USA90:5428-5432 (1993)). Briefly, RNA was dephosphorylated using bacterialalkaline phosphatase (Gibco BRL, Gaithersberg, Md.) and end-labeled atthe 5′ with T4 polynucleotide kinase (New England Biolabs, Beverly,Mass.) and [γ³²P]ATP (Amersham Pharmacia Biotech, Piscataway, N.J.)(Fitzwater et al, Methods Enzymol. 267:275-301 (1996)). Direct bindingwas performed by incubating ³²P-RNA with purified platelets in plateletcounts ranging 100,000 to 97/μL in Tyrodes buffer+1 mg/ml BSA at 37° C.The fraction bound of the nucleic acid-protein complex was quantifiedwith a Phosphoimager (Molecular Dynamics, Sunnyvale, Calif.). Thenon-specific binding of radiolabeled nucleic acid was subtracted (Wonget al, Proc. Natl. Acad. Sci. USA 90:5428-5432 (1993)).

RNA binding to gpIIb/IIIa. To measure aptamer binding to gpIIb/IIIa, RNAwas 5′-biotinylated and assayed in an enzyme linked oligonucleotideassay (ELONA) (Drolet et al, Nat. Biotechnol. 14:1021-1025 (1996)).Briefly, biotin was appended to the 5′ end of the RNA by standardtranscription protocols using 4-fold molar excess of 5′-biotin GMP overGTP in the reaction mixture. Immulon 2 wells were coated overnight at 4°C. with gpIIb/IIIa. The wells were washed and blocked with 3% bovineserum albumin (BSA) for 1 h at room temperature. Two-fold serialdilutions of RNA from 1 μM to 980 ρM were performed and the RNA wasincubated in the protein-coated well at 37° C. for 45 min. Unbound RNAwas removed by washing. To detect bound RNA, 1:1000streptavidin-alkaline phosphate conjugate (Sigma-Aldrich Corp., St.Louis, Mo.) was incubated in the wells for 30 min at room temp. Finallyp-nitrophenyl phosphate (Sigma-Aldrich Corp., St. Louis, Mo.) was usedas a substrate and, after addition, absorbance at 405 nm was measuredevery 30 sec over 30 min in a EL311 Microplate Autoreader (Bio-tekInstruments, Inc., Winooski, Vt.). Binding data is fit to an equationthat describes the fraction of RNA bound as a function of K_(d) formonophasic binding behavior.

Competition Assay

The assay was carried out as above with the exception that afteraddition of 5′-biotinylated RNA, either a) buffer, b) cold (unlabeledwith ³²P) gpIIb/IIIa RNA, c) Abciximab (Eli Lilly, Indianapolis, Ind.)or d) Eptifibatide (COR Therapeutics Inc, San Fran Francisco, Calif.)was added at two-fold serial dilutions between 100 to 0.1-fold excess ofthe compound's dissociation constant.

Functional Assays

Platelet Function Analysis (PFA). Platelet Function Analyzer, PFA-100(Dade Behring, Deerfield, Ill.) provides a quantitative measure ofplatelet function in anti-coagulated whole blood (Ortel et al, Thromb.Haemost. 84:93-97 (2000)). Briefly, 800 μL of whole blood was mixed withaptamers in a platelet binding buffer consisting of 150 mM NaCl; 20 mMHEPES pH: 7.4; 5 mM KCl; 1 mM MgCl₂ and CaCl₂. The maximum closing timeof the PFA-100 is 300 seconds. Antidote activity of aptamer was measuredby mixing whole blood with aptamer in buffer followed by administrationof antidote and measuring in PFA.

Platelet Aggregometry. Chrono-log Whole Blood Lumi Ionized Aggregometer(Chrono-log, Haverton, Pa.) provided a measurement of plateletaggregation in platelet-rich plasma. Briefly, platelet-rich plasma (PRP)was isolated from whole blood and 450 μl of PRP, 50 μl of aptamer and 50μl Chono-lume were added. After calibrating the instrument, 5 μl of ADPagonist was added and transmission was measured for 6 minutes.

Results

A solid phase platform of SELEX was utilized whereby the protein wasadsorbed to plates and the presence and integrity of the protein wasverified by ELISA. In this assay, two antibodies were used, CD41, whichrecognized the gpIIb/IIIa complex, and CD61, which recognizes the β₃subunit of the heterodimer. Ethylene diamine tetra-acetic acid (EDTA), acalcium chelator, was used to demonstrate the confirmation-specificnature of gpIIb/IIIa. It was clear that both human and porcinegpIIb/IIIa on the plates were in a confirmation that was recognized byboth antibodies without EDTA.

After determining that the protein was adsorbed to the plates and wasrecognized by both complex- and monomer-specific antibodies, a ‘toggle’selection was performed in order to isolate RNA ligands that bound toboth human and porcine orthologs (Ginsberg et al, Hematology (1):339-357(2001)). The selection was monitored using real-time PCR as described(Lupoid et al, Cancer Research 62:4029-4033 (2002)). As illustrated inTable 1, the signal from the enrichment from the gpIIb/IIIa wells wasabove that of the no protein well. At round 12, there was a 113-foldincrease in the signal to background and this was interpreted torepresent a significant enrichment of the RNA pool to gpIIb/IIIa.Subsequent rounds of selection resulted in a significantly reducedsignal to background (data not shown), and at this point, round 12 wascloned and sequenced.

TABLE 1 Absolute RNA Round Protein No protein Signal/Background 1 — — —2 — — — 3 2.45E+00 2.50E+01 0.1 4 2.12E+01 7.93E+00 3 5 1.70E+021.05E+02 2 6 7.98E+01 1.59E+01 5 7 1.29E+02 9.41E+01 1 8 9.95E+011.48E+01 7 9 5.44E+00 2.31E+00 2 10 5.63E+01 3.51E+00 16 11 1.56E+022.32E+01 7 12 3.72E+01 3.28E−01 113

In order to correlate the real-time data with binding affinity, purified11D platelets were isolated and the affinity of each round to activatedplatelets was measured using nitrocellulose-filter partitioning (Wang etal, Biochemistry 32:1899-1904 (1993)). Since purified gpIIb/IIIa wereselected, 80,000 gpIIb/IIIa receptors per platelet were assumed (Tcheng,Am. Heart J. 139:S38-45 (2000)) and the theoretical concentration of theprotein was calculated. While this certainly does not provide anaccurate binding affinity, it was useful to validate the real-time PCRdata. The binding data illustrated the increased affinity of the roundsto gpIIb/IIIa on platelets (FIG. 1). Moreover, the binding alsocorrelated with signal:background data in Table 1, where round 12 boundto the platelets with the highest affinity (FIG. 1) and represented thehighest signal:background in the selection.

The resulting clones from round 12 were clustered into 10 distinctfamilies (Table 2). Representative clones from each family weresubsequently tested for their ability to bind to purified platelets.Aptamer C5 had the highest affinity interaction with gpIIb/IIIa onplatelets (apparent K_(d)=2 nM). Clone C1, which was the highestrepresented clone from round 12, had an apparent K_(d)=6 nM. Clone C3was the poorest binder to gpIIb/IIIa, with an apparent K_(d)=62 nM (FIG.2).

TABLE 2 Clone Sequence of N40 region SEQ ID NO: Frequency C1TATAGACCACAGCCTGAGTATTAACCACCAACCCAGGTACT 1 51% C2TATAACCGTTCTAGCGCTAATGACACTATAGCATCCCCGT- 2  2% C3TGCCACATGCCTCAGATACAGCACGCACCTTCGACCTAAT- 3 12% C4ACCTGCTAGCAGTGGCGCGAATAAACCATCGCAGCATCAA- 4  2% C5GGACTTGCGAGCCAGTCCACACGCCGCGACTAAAGAGACTTCTC 5  2% C6ACAGATCTACCCGAGACAAACATCCCACCCTCCGA------ 6  7% C7TCCTAAGATTAAATACGCCACGGCTCACTTACACACCAG-- 7  4% C8TGCCACATGCCTCAGATACAGCACGCACCTTCGACCTAAT- 8 12% C9TCCCTTGGATGAGACTAACAACCTACCACATCCTA-TACTC 9  4%

In order to assess the inhibitory activity of the aptamer ongpIIb/IIIa-mediated platelet aggregation, each aptamer was tested in aPlatelet Function Analyzer (PFA-100). This device is sensitive togpIIb/IIIa-mediated platelet inhibition with Abciximab and Eptifibatide(data not shown) (Hezard et al, Thromb. Haemost. 81:869-873 (1999)) andis an attractive assay as it measures platelet activity in whole bloodunder high shear conditions, which recapitulates the in vivo conditionmore reasonably than standard aggregometry (Harrison, Blood Rev.19:111-123 (2005)). In addition to the clones isolated from theselection, an RNA aptamer generated to gpVb/IIIa, a related integrin togpIIb/IIIa, designated Cl, was also tested. All the clones were testedin a volume of 840 μL at a final concentration of 1 μM (FIG. 3A). Thebaseline closing time of human whole blood was 95±1 s. Of the clonestested in human whole blood, Cl was the only aptamer that inhibitedplatelet aggregation to >300 s, exceeding the upper limit of theinstrument. Given the binding data of the aptamers to platelets, theconclusion was that the aptamers isolated to gpIIb/IIIa bound to theligand on platelets without affecting its function upon activation. Anevaluation was then made of the ability of the aptamers to inhibitplatelet function in pig blood to see if any of the isolated ligands hadany effect. Not surprisingly, none of the aptamers tested had any effectin the PFA-100 and modestly deviated from the baseline closing time91±15 s without significance (FIG. 3B).

Finally, after determining the effect of Cl in PFA, a determination ofthe platelet activity was made in a more traditional assay. As shown inFIG. 3C, Cl was tested in a Chrono-log lumi-aggregometer. Transmittanceof the negative control was 104±2%, while Cl was 5%. This was equivalentto Eptifibatide, which served as a positive control.

In order to determine the binding affinity of Cl to gpIIb/IIIa, Cl waslabeled with biotin at its 5′-end and bound to gpIIb/IIIa immobilized onplates (Drolet et al, Nat. Biotechnol. 14:1021-1025 (1996)), with aK_(d) of 10±5 nM. To determine the binding region of Cl, the aptamer wasthen analyzed in a competition assay against Abciximab and Eptifibatideover a concentration range between 0.1- and 100-fold excess of the K_(d)of each drug (FIG. 4). Both gpIIb/IIIa blockers competed with aptamer Clin a concentration-dependent manner.

After establishing that Cl inhibited platelet aggregation in vitro, theactivity of truncated versions of the molecule was assessed. It wasdetermined that a modestly truncated version, designed Cl-6, was just aspotent in inhibiting platelet aggregation, exceeding the closing time of300 at a concentration of 500 nM. This level of inhibition was withinthe same range as Eptifibatide, which is extensively used in the clinic(Jackson et al, Nat. Rev. Drug Discov. 2:775-789 (2003)).

Once a concentration of Cl-6 that increased the closing time to >300 shad been identified, 5 antidote oligonucleotides (AO) were designed thatwere the reverse compliment of a segment of previous random region ofCl-6 (FIG. 5A). The AO were added at 10-fold molar excess of Cl-6 (AO 5μM versus Cl-6 500 nM). Each antidote effectively reversed the activityof Cl-6 and their closing time was similar to baseline (P>0.05) (FIG.5B). The most effective antidote, AO2, had a closing time of 81±19 s,while the least effective antidote, AO5, had a closing time of 130±15 s.A scrambled AO (AOSc) was used to verify that the reversal activity wasspecific to each antidote and not a consequence of the presence ofadditional nucleic acid in the assay. When mixed with Cl-6, AOScresulted in a closing time of >300 s (FIG. 5B).

In summary, a solid-phase system of SELEX was employed to isolate2′-fluoropyrimidine modified RNA aptamers that bound to gpIIb/IIIa withhigh affinity (FIG. 2). The aptamer with the highest affinity, C5, boundto gpIIb/IIIa on the surface of platelets with a K_(d) of 2 nM. Inevaluating gpIIb/IIIa-mediated platelet aggregation in whole blood, itwas demonstrated that aptamer Cl exceeded the upper limit of the assaywith a closing time>300 s in human blood. A truncated version of thisaptamer, Cl-6, retained inhibitory activity in the PFA-100 assay. It wasinteresting that the other aptamers isolated to gpIIb/IIIa did not haveinhibitory activity despite high affinity binding to the protein. It ispossible the protein immobilized on the solid surface was in aconformation that prevented RNA ligand access to its functional epitope.Aptamer Cl did not have the highest affinity to gpIIb/IIIa yet was theonly one with significant functional activity. Eptifibatide isillustrative of this, with a K_(d) of 120 nM, compared to Abciximab,which has a K_(d) of 5 nM (Scarborough et al, Circulation 100:437-444(1999)).

After establishing the inhibitory effect of Cl, its binding wascharacterized. Not surprisingly, both Eptifibatide and Abciximab competewith the aptamer for binding to gpIIb/IIIa (FIG. 4). Abciximab is ahumanized mouse Fab (also known as 7E3) that binds to both gpIIb/IIIaand gpVb/IIIa (Artoni et al, Proc. Natl. Acad. Sci. USA 101:13114-13120(2004)). Binding analysis has shown that this antibody preferentiallybinds to active platelets over resting ones and its effect on gpIIb/IIIainhibition is its interaction with the β₃ subunit (Artoni et al, Proc.Natl. Acad. Sci. USA 101:13114-13120 (2004)). Eptifibatide is cyclicheptapeptide modeled after a leucine-glycine-aspartic acid (KGD)sequence from pit viper venom (Coller, Thromb. Haemost. 86:427-443(2001)). Its inhibitory action is on the arginine-glycine-aspardic acid(RGD) residue on gpIIb/IIIa (Scarborough et al, Circulation 100:437-444(1999)). The RGD moiety binds to both gpIIb/IIIa and gpVb/IIIa as welland involves the β₃ subunit (Xiong et al, Science 296:151-155 (2002),Xiao et al, Nature 432:59-67 (2004)) and, therefore, either the aptameris sterically hindering fibrinogen from accessing the RGD pocket betweenthe α₂ and β₃ pocket or preventing the receptor from forming theconformation necessary for fibrinogen binding.

All of the antidote oligonucleotides to Cl-6 functionally reversed theactivity of the aptamer, returning the closing times to baseline levels(FIG. 5B). Rational design of AO is based on the assumption that thetertiary conformation of the aptamer can be disturbed by Watson-Crickbase-pairing of AO to critical regions of the aptamer (Rusconi et al,Nat. Biotechnol. 22:1423-1428 (2004), Rusconi et al, Nature 419:90-94(2002)). There was insignificant variability between AOs as they alleffectively inhibited the aptamer's anti-platelet activity. It wasremarkable to see the rate of the aptamer-antidote binding. Afterincubation of the aptamer for 1 min, the antidote is added, carefullymixed and then tested. This fast aptamer-antidote interaction is veryattractive as a regulatable therapeutic as it gives the clinician tightcontrol of the anti-platelet agent and the ability to reverse itsactivity immediately in the event of a complication requiring normalplatelet activity.

This anti-gpIIb/IIIa aptamer/antidote represents the first regulatableanti-platelet drug/antidote pair that has the potential to significantlyimprove morbidity in patients that require gpIIb/IIIa inhibitors.

EXAMPLE 2

Over the past decade, much research has elucidated the important role ofplatelets in cardiovascular disease. Excessive accumulation of plateletson atherosclerotic plaques is an essential aspect of thrombus formation,which, in turn, is responsible for the development of acute coronarysyndromes like stroke and arterial thrombosis. A number of anti-plateletdrugs exist that are routinely used in clinics. Aspirin inhibitsthromboxane A2 and was the first anti-platelet agent used clinically.Clopidogrel and Ticlopidine inhibit ADP receptors PIIY1 and PIIY12 andAbciximab, Eptifibatide and Tirofiban are gpIIb/IIIa inhibitors, themost potent class of anti-platelet compounds to date. While these drugshave shown remarkable clinical efficiency in reducing the morbidity andmortality associated with thrombosis, these agents have a number ofdrawbacks, most significant of which is hemorrhage. Therefore, apressing need exists for anti-platelet drugs with improved safetyprofiles that are targeted against a platelet receptor/ligandinteraction involved in the common platelet activation pathway. Antidotedevelopment represents a key strategy to overcome the obstacle ofhemorrhage and, in order to address this issue for anti-platelettherapies, von Willebrand Factor (VWF) inhibitors have been developedthat have specific antidotes.

Using the SELEX technique, aptamers were isolated from a2′-fluoropyrimidine-modified single-stranded RNA library containing a 40nucleotide-randomized region that bind to VWF with high affinity andspecificity. Employing the SELEX procedure yielded aptamers rapidly andmade it possible to assess the inhibitory function in in vitroexperiments. Previously, nuclease-resistant aptamers have been isolatedthat bind to and inhibit human factors Vila, IXa, Xa and IIa using“SELEX”. As with numerous selection experiments previously conducted,nitrocellulose-filter binding was employed as the partitioning scheme.Briefly, ³²P-end-labeled RNA aptamers (<0.1 nM) were incubated with theindividual protein at a range of concentrations. The RNA-proteincomplexes were separated from the free RNA by passing the mixturethrough a nitrocellulose filter by vacuum. Bound and free RNA werequantified by phosphorimager analysis and the data fitted to yield theK_(d)s for the RNA aptamer-protein interaction. A decreasing K_(d) valuepointed to increasing affinity of RNA molecules for VWF. The RNA roundthat yielded a binding affinity in low nanomolar range is sequenced andindividual clones are grouped into families based on their sequencesimilarity and structural conservation using computer-aided secondarystructure analysis.

VWF “SELEX”: Using the starting library, 9 rounds of selection wereperformed to purified human VWF protein (obtained from Haemtech Inc.).There was a steady increase in binding affinity to VWF from the startinglibrary to R9 (VWF selection round 9) (FIG. 6.). The Kd of round 9reached the single digit nanomolar range, thus the individual clonesmaking up the R9RNA pool were cloned and characterized. (See FIGS. 7 and8.)

Clone VWF R9.14 (SEQ ID NO: 10) GGGAGGACGATGCGG-

CAGACGACTCGCTGAGGATCC

Binding Affinities

Clone VWF R9.14 Kd=12 nM

SO VWF AO 1 (SEQ ID NO: 11)mC.mU.mU.mA.mA.mG.mC.mA.mG.mG.mA.mG.mA.mG.mC.mG. mC.mG.mA.mU SO VWF AO2(SEQ ID NO: 12) mA.mG.mC.mU.mG.mC.mU.mU.mA.mA.mG.mC.mA.mG.mG.mA.mG.mA.mG.mC SO VWF AO3 (SEQ ID NO: 13)mU.mU.mG.mA.mU.mA.mG.mC.mU.mG.mC.mU.mU.mA.mA.mG. mC.mA.mG.mG SO VWF AO4(SEQ ID NO: 14) mG.mC.mU.mA.mU.mU.mU.mG.mA.mU.mA.mG.mC.mU.mG.mC.mU.mU.mA.mA SO VWF AO5 (SEQ ID NO: 15)mA.mA.mG.mA.mU.mG.mG.mG.mC.mU.mA.mU.mU.mU.mG.mA. mU.mA.mG.mC.mU.mGSel2 3′ W1 (SEQ ID NO: 16)mT.mC.mT.mC.mG.mG.mA.mT.mC.mC.mT.mC.mA.mG.mC.mG. mA.mG.mT.mC.mG.mT.mC.mT

EXAMPLE 3

To generate a safer, antidote-controllable VWF inhibitor, the decisionwas made to exploit the properties of nucleic acid ligands termedaptamers. As noted above, aptamers are single-stranded nucleic acidmolecules that can directly inhibit protein function by binding to theirtargets with high affinity and specificity (Nimjee, Rusconi et al,Trends Cardiovasc. Med. 15:41-45 (2005)). To isolate RNA aptamersagainst VWF, a modified version of SELEX (Systematic Evolution ofLigands by EXponential enrichment), termed “convergent” SELEX, wasperformed. These aptamers bind to VWF with high affinity (K_(d)<20 nM)and inhibit platelet aggregation in Platelet Function Analyzer (PFA-100)and ristocetin induced platelet aggregation (RIPA) assays. Moreover, anantidote molecule that can quickly reverse such aptamers' function hasbeen nationally designed. This antidote molecule can give physiciansbetter control in clinics, enhancing the aptamers' safety profile.

Experimental Details

Generation of Aptamers

“Convergent” SELEX

The sequence of the starting RNA combinatorial library was5′-GGGAGGACGATGCGG-N₄₀-CAGACGACTCGCTGAGGATCC-3′ (SEQ ID NO: 17), whereN₄₀ represents 40 completely random nucleotides. 2′F cytidinetriphosphate and 2′F uridine triphosphate (Trilink Biotechnologies, SanDiego, CA) were incorporated into the RNA libraries by in vitrotranscription in order to confer nuclease resistance. The selection wascarried out in selection buffer E (20 mM HEPES, pH 7.4, 50 mM NaCI, 2 mMCaCl₂, and 0.1% bovine serum albumin (BSA)) at 37° C. until round P5V2and then continued in selection buffer F (20 mM HEPES, pH 7.4, 150 mMNaCI, 2 mM CaCl₂, and 0.1% bovine serum albumin (BSA)). RNA-VWFcomplexes were separated from unbound RNA by passing them over anitrocellulose filter (BA 85, Whatman Inc, NJ).

Five rounds of SELEX were performed on the plasma proteome followed byfour rounds of convergent SELEX as described by Layzer et al.(oligonucleotides 17:XX-XX (2007)). Briefly, the starting aptamerlibrary (Sel2) was incubated with diluted normal human plasma at 37° C.for 15 minutes in selection buffer E. Yeast tRNA was used to inhibitnon-specific binding of the aptamer library to the plasma proteome.Bound RNA aptamers were separated from unbound aptamers using anitrocellulose filter. Following round 5 of plasma SELEX, convergentSELEX using VWF was performed for 4 rounds (2 rounds in selection bufferE followed by 2 rounds in selection buffer F).

Antidote Oligonucleotides

Antidote oligonucleotides were synthesized and purified by DharmaconResearch, Inc. 2′-O-methyl purines and pyrimidines were incorporatedinto the antidote oligonucleotides.

Binding Assays

Affinity constants (K_(d) values) were determined using double-filternitrocellulose filter binding assays (Rusconi et al, Thromb. Haemost.84:841-848 (2000)). All binding studies were performed in either bindingbuffer E (20 mM HEPES, pH 7.4, 50 mM NaCl, 2 mM CaCl₂, and 0.1% BSA) orbinding buffer F (20 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM CaCl₂, and 0.1%BSA) at 37° C. Human purified VWF (factor VIII free) was purchased fromHaematologic Technologies Inc. (Essex Junction, VT) and used in thedouble-filter nitrocellulose filter binding assay to determine the K_(d)of every other round and individual clones. VWF SPI and VWF SPIIIdomains were kindly provided by Dr. J. Evan Sadler (WashingtonUniversity in St. Louis). Briefly, RNA were dephosphorylated usingbacterial alkaline phosphatase (Gibco BRL, Gaithberg, Md.) andend-labeled at the 5′ end with T4 polynucleotide kinase (New EnglandBiolabs, Beverly, Mass.) and [γ³²P] ATP (Amersham Pharmacia Biotech,Piscataway, N.J.) (Fitzwater and Polisky, Methods Enzymol. 267:275-301(1996)). Direct binding was performed by incubating ³²P-RNA with VWF inphysiological buffer+1 mg/ml BSA at 37° C. for 5 min. The fraction ofthe nucleic acid-protein complex which bound to the nitrocellulosemembrane was quantified with a posphoimager (Molecular Dynamics,Sunnyvale, Calif.). Non-specific binding of the radiolabeled nucleicacid was subtracted out of the binding such that only specific bindingremained (Wong and Lohman, Proc. Natl. Acad. Sci. USA 90:5428-5432(1993)).

Platelet Function Analysis

PFA-100

The Platelet Function Analyzer, PFA-100 (Dade Behring, Deerfield, Ill.),measures platelet function in terms of clot formation time. In thisassay, collagen/ADP cartridges were utilized to activate the plateletsand measure the amount of time taken to form a clot in anticoagulatedwhole blood (Harrison, Blood Rev. 19:111-123 (2005)). Briefly, 840 μL ofwhole blood was mixed with aptamer in platelet binding buffer (150 mMNaCl; 20 mM Hepes pH: 7.4; 5 mM KCl; 1 mM MgCl₂ and 1 mM CaCl₂) andincubated for 5 minutes at room temperature. This mixture was then addedto a collagen/ADP cartridge and tested for its closing time. The maximumclosing time of the PFA-100 is 300 seconds. Antidote activity of theaptamer was measured by mixing whole blood with aptamer, incubating for5 minutes followed by addition of antidote or buffer, and testing themixture in the PFA-100.

Platelet Aggregometry

A Chrono-log Whole Blood Lumi Ionized Aggregometer (Chrono-log,Haverton, Pa.) was used to provide a measurement of platelet aggregationin platelet-rich plasma. Briefly, platelet-rich plasma (PRP) wasisolated from whole blood collected in 3.2% buffered trisodium citratetubes (BD Vacutainer Systems, Franklin Lakes, N.J.); aptamer was addedand incubated with the blood for 5 minutes before testing. Aftercalibrating the instrument, 5 μL of agonist was added and transmissionwas measured for 10 minutes.

Ristocetin-Induced Platelet Aggregation (RIPA)

Ristocetin-induced platelet aggregation was performed using plateletrich plasma (PRP) from healthy volunteers. Clone VWF R9.3 or VWF R9.14was mixed with 400 μL of PRP in a flat bottom glass tube; ristocetin(Helena Laboratories, TX) was added to a final concentration of 1.25mg/mL. The PRP was stirred using a steel stir bar at 37° C. andturbidity was monitored as percent light transmitted for 10 minutes.

Collagen-Induced Platelet Aggregation (CIPA)

Collagen-induced platelet aggregation was performed using platelet richplasma (PRP) from healthy volunteers. Clone VWF R9.3 or VWF R9.14 wasmixed with 400 μL of PRP in a flat bottom glass tube and collagen wasadded to a final concentration of 2 μg/mL. The PRP was stirred using asteel stir bar at 37° C. and turbidity was monitored as percent lighttransmitted for 10 minutes.

ADP-Induced Platelet Aggregation (AIPA)

ADP-induced platelet aggregation was performed using platelet richplasma (PRP) from healthy volunteers. Clone VWF R9.3 or VWF R9.14 wasmixed with 400 μL of PRP in a flat bottom glass tube and ADP was addedto a final concentration of 10 uM. The PRP was stirred using a steelstir bar at 37° C. and turbidity was monitored as percent lighttransmitted for 6 minutes.

Thrombin-Induced Platelet Aggregation (TIPA)

Thrombin-induced platelet aggregation was performed using platelet richplasma (PRP) from healthy volunteers and SFLLRN peptide. Clone VWF R9.3or VWF R9.14 was mixed with 400 μL of PRP in a flat bottom glass tubeand SFLLRN was added to a final concentration of 2 nM. The PRP wasstirred using a steel stir bar at 37° C. and turbidity was monitored aspercent light transmitted for 6 minutes.

Results

Five Rounds SELEX Followed by Four Rounds of “Convergent” SELEX YieldedAptamers that Bind to VWF with High Affinity.

To isolate RNA aptamers against VWF, a modified version of SELEX(Systematic Evolution of Ligands by EXponential enrichment) wasperformed. First, an RNA library containing 2′-fluoropyrimidines wasincubated with total plasma proteins; the RNA ligands that bound to thisproteome were recovered. Four additional rounds of SELEX were performedagainst the plasma proteome to generate a focused library that washighly enriched for RNA ligands that bound plasma proteins. Next,convergent SELEX (Layzer, Oligonucleotides 17:XX-XX (2007)) wasperformed to isolate those RNA aptamers from the focused library thatspecifically bound VWF. Such convergent SELEX progressed rapidly; theaffinity of the round 4 pool of aptamers had a K_(d) of 4.5 nM for VWF(FIG. 9A). Next, the identity of the individual aptamers was determinedby cloning and sequencing. As shown in Table 3, two sequences dominatedfollowing round 4 of convergent SELEX against VWF (clones 9.3 and 9.4).These dominant clones, along with two less abundant clones (9.18 and9.14), were chosen for further evaluation. To characterize the bindingof these aptamers to VWF, nitrocellulose filter binding assays wereperformed. As shown in Table 3 and FIG. 1B, three of the four clones(9.3, 9.4 and 9.14) bound to VWF with high affinity (K_(d)<20 nM) (FIG.1B). Thus, by performing 5 rounds of SELEX on the plasma proteomefollowed by 4 rounds of convergent SELEX against VWF using the plasmaproteome focused library, aptamers were identified that bound to VWFwith high affinity.

TABLE 3 P5V4 Aptamer Sequences Clone ID Variable Region SequenceSEQ ID NO: Frequency (%) Kd VWF R9.35′-ATCGCGCTCTCCTGCTTAAGCAGCTATCAAATAGCCCACT-3′ 18 39 1.2 nM VWF R9.45′-TATAGACCACAGCCTGAGATTAACCACCAACCCAGGACT-3′ 19 36 1.9 nM VWF 89.185′-TGCTCCTTGGCCTTAGCCCTGGAACCATCAATCCTCTTCG-3′ 20 3 278 nM VWF R9.145′-TGGACGAACTGCCCTCAGCTACTTTCATGTTGCTGACGCA-3′ 21 1  12 nM VWF R9.905′-ACGNGTANACCTGCTACAATANCAGCCTAAATGGCCCACT-3′ 22 1 N/D VWF R9.665′-ATCCCTGCCAAACATACTTTCGCTTTGGCTAGGACTCCCT-3|′ 23 3 N/D VWF R9.375′-GCACCCCCTCGACAACGACCCTGTGCCCCTCGATCGACCA-3′ 24 2 N/D VWF R9.545′-CCCATTACGGCTT-CCTTGTATTCTTGGACAAGCCGCGACT-3′ 25 2 N/D VWF R9.355′-ACCCTTGACAACAACCCTTCCTCACCAACCCCTCCCAAC-3′ 26 1 N/D VWF R9.815′-ATACCCTCGACAACGACCCTATTCGCATGACACCTCTGTG-3′ 27 1 N/D VWF R9.335′-ATGAATCCTCCTGTCGAACAACAGCTGTTTCAGCCCAACT-3′ 28 1 N/D VWF R9.935′-GACCGACTGATTCGCACCAGACCACGACGTTATGGCCCAA-3′ 29 1 N/D VWF R9.745′-GTCGACTTAGCCCCGTGCTCGGCGCTTCACAGTCGACTAT-3′ 30 1 N/D VWF R9.415′-CGAGATCACACTGCCCCAATAGCCACTGAACTAGCGCGCA-3′ 31 1 N/D VWF R9.465′-ACCATTCGCGAGCACAACGCTTTGTACTCAACACTCCACG-3′ 32 1 N/D VWF R9.495′-ACCGTTCAGAAATGACCCCACGCACATCCATCCCTGAGCT-3′ 33 1 N/D VWF R9.975′-ACGTGATCCTCGGACCCAGCATTGCATTATATGCGCCCCT-3′ 34 1 N/D VWF R9.955′-ACTCTCAGCCCATGTGCCTCAACCAAGGCACGGCTTGCTC-3′ 35 1 N/D VWF R9.625′-CACCCTTCACCCGAACCCTGCCC-ACG-ACCCCACACCCCGC-3′ 36 1 N/D VWF R9.575′-ATGACCAGCCCCTCGACAACGACCCTGCTGGCTCAACCGTT-3′ 37 1 N/D VWF 89.1185′-GACCGCCGCNNCCGACCCNAGNNNTGCTGTGTNCGCTCCGCC-3′ 38 1 N/D N/D-notdeterminedClones VWF R9.3 and VWF R9.4 Bind to the VWF SPIII Domain but not to theVWF SPI Domain; Clone VWF R9.4 Binds to Both the VWF SPI and SPIIIDomains.

To determine the specific binding domains of selected aptamer clones onVWF, studies were performed using VWF SPI and VWF SPIII domains. SP Iand SP III are V8 protease fragments of VWF from the N-terminus of theprotein. SPIII is 1365 residues in length (aa 1-1365) containing domainsfrom D′ mid-way through D4, including the A1 domain. SPI represents theC-terminal 455 residues of SPIII and contains mainly domain A3 and apart of domain D4 (FIG. 1D).

Clones VWF R9.3 and VWF R9.14 bound to the SPIII fragment but not to theSPI fragment (FIG. 1C and FIG. 1D). These results suggest that theseaptamers bind proximal to the positively charged A1 domain of VWF. TheA1 domain is mainly involved in platelet aggregation since it makes thecontact with the GP Ibα subunit of platelet receptor GP Ib-IX-V. CloneVWF R9.4 bound to both SPI and SPIII domains, mapping its bindingproximal to the VWF A3 domain (FIG. 1C).

Clones VWF R9.3 and VWF R9.14 but not VWF R9.4 Inhibited PlateletFunction Measured by PFA-100.

To determine whether the isolated aptamers had any effect on plateletactivity, they were evaluated for their ability to limitplatelet-induced clot formation in a PFA-100 assay. The PFA-100instrument uses small membranes coated with collagen/ADP orcollagen/epinephrine to screen for the presence of platelet functionaldefects. As shown in FIG. 10A, VWF aptamers R9.3 and R9.14 inhibitedplatelet dependent clot formation completely in the PFA-100 assay(closing time>300 s) at a concentration of 1 μM. In contrast, VWFaptamer R9.4, while having a K_(d) similar to R9.3 and R9.14, had noactivity (FIG. 10A). Next, to determine the minimum effective dose ofVWF aptamer R9.3 and VWF aptamer R9.14, a dose titration study wasperformed. As shown in FIG. 10B, both aptamers completely inhibitedplatelet function (CT>300 s) at concentrations greater than 40 nM innormal whole blood in the PFA-100 assay (FIG. 10B). Thus, atconcentrations above 40 nM, these two aptamers inhibit platelet functionto the level seen in patients with severe VWD.

Clones VWF R9.3 and VWF R9.14 Inhibited Platelet Aggregation Measured byRIPA but Not with CIPA, AIPA and TIPA.

To confirm these findings and to determine the specificity of the VWFaptamers, platelet aggregation studies were performed. First, aninvestigation was made of the effects of VWF aptamers R9.3 and R9.14 ina ristocetin induced platelet aggregation (RIPA) assay to determine ifthe aptamers inhibit platelet function by blocking VWF's ability tointeract with GP Ib-IX-V. Ristocetin was used as a VWF antagonistbecause it binds specifically to VWF in platelet rich plasma (PRP) andassists in VWF-mediated platelet activation/aggregation through the GPIb-IX-V receptor. Other antagonists (collagen, ADP and thrombin) thatactivate platelets through pathways that are not dependent on the VWF-GPIb-IX-V interaction were also evaluated to determine if the aptamers hadany inhibitory effect on these additional activation pathways. As shownin FIG. 10C, VWF aptamers R9.3 and R9.14 completely inhibited RIPA (at aconcentration of 250 nM), illustrating that the aptamers can potentlyinhibit the VWF-GP Ib-IX-V interaction. In contrast, the aptamers had noeffect in collagen, ADP or thrombin induced platelet aggregation (FIG.10C). Thus, VWF aptamers R9.3 and R9.14 inhibit platelet function byspecifically blocking VWF-GP Ib-IX-V-mediated platelet activation andaggregation.

Antidote Oligonucleotide 6 (AO6) can Reverse VWF R9.14 Binding to VWF toBackground Levels.

Six different antidote oligonucleotides (AO1-6) were designed to bind toVWF aptamer R9.14 through Watson-Crick base pairing rules (FIG. 11A).This strategy has been successfully employed to design an antidote tocontrol the activity of an aptamer to factor IXa (Rusconi et al, Nature419:90-94 (2002), Rusconi et al, Nat. Biotechnol. 22:1423-1428 (2004),Nimjee et al, Mol. Ther. 14:408-415 (2006)). To determine if theantidote oligonucleotides could inhibit aptamer binding to VWF, theywere evaluated in a nitrocellulose filter binding assay. As shown inFIG. 11B, the most effective antidote for VWF aptamer. R9.14 is AO6.This antidote can reverse VWF aptamer R9.14's ability to bind VWF tobackground levels (FIG. 11B).

AO6 Can Reverse the Effects of VWF R9.14 Completely in a PFA-100 Assay.

Since AO6 can reverse VWF aptamer 9.14 binding to VWF, it was nextdetermined whether the antidote could also reverse the aptamer'sactivity in a whole blood clinical lab assay was tested. To that end,the ability of AO6 to inhibit VWF aptamer 9.14 was tested in a PFA-100assay. As shown in FIG. 12A, the antidote can reverse the activity ofthe aptamer in a dose dependent manner. Moreover, the antidote is ableto completely reverse the antiplatelet effects of the VWF aptamer R9.14at a 40-fold excess of aptamer concentration. In contrast, a scrambledversion of the antidote oligonucleotide (Scr AO6) had no effect onaptamer activity (FIG. 12A). Thus, antidote AO6 is able to restoreplatelet function in a whole blood assay back to normal levels, even inthe presence of enough VWF aptamer 9.14 (40 nM) to impede plateletfunction to an extent consistent with VWD.

AO6 Can Quickly Reverse the Effects of VWF R9.14 for a Sustained Periodof Time in a PFA-100 Assay.

For such an antidote to be useful clinically, the antidote should beable to act quickly and for a prolonged period of time. To determine howrapidly AO6 could reverse the aptamer and how long such reversal issustained, a time course assay was performed using the PFA-100. As shownin FIG. 12B, AO6 can rapidly reverse the effects of VWF aptamer R9.14 inless than 2 minutes. Moreover, once the antiplatelet activity isreversed, the antidote maintained its ability to sustainably inhibit theaptamer for greater than 4 hours (FIG. 12C). AO activity could not betested for more than 4 hours due to platelet degradation over such time.These results demonstrate that AO6 can rapidly and durably reverse theeffects of VWF aptamer R9.14.

In summary, aptamers are single-stranded nucleic acid molecules that candirectly inhibit protein function by binding to their target with highaffinity and specificity. To date, a number of proteins involved incoagulation have been targeted by aptamers, successfully yieldinganticoagulant molecules with therapeutic potential (Rusconi et al,Thromb. Haemost. 84:841-848 (2000), Rusconi et al, Nature 419:90-94(2002), Becker et al, Thromb. Haemost. 93:1014-1020 (2005), Nimjee etal, Trends Cardiovasc. Med. 15:41-45 (2005)). Aptamers represent anattractive class of therapeutic compounds for numerous reasons. They arerelatively small (8 kDa to 15 kDa) synthetic compounds that possess highaffinity and specificity for their target proteins (equilibriumdissociation constants ranging from 0.05-40 nM). Thus, they embody theaffinity properties of monoclonal antibodies with the chemicalproduction properties of small peptides. In addition, preclinical andclinical studies to date have shown that aptamers and compounds ofsimilar composition are well tolerated, exhibit low or noimmunogenicity, and are suitable for repeated administration astherapeutic compounds (Dyke et al, Circulation 114:2490-2497 (2006)).Moreover, bioavailability and clearance mechanisms of aptamers can berationally altered by molecular modifications to the ligand (i.e.cholesterol or polyethylene glycol). Most importantly, it has been shownthat antidote oligonucleotides can be rationally designed that negatethe effect of aptamers in vitro and in vivo (Rusconi et al, Nature419:90-94 (2002), Rusconi et al, Nat. Biotechnol. 22:1423-1428 (2004),Nimjee et al, Mol. Ther. 14:408-415 (2006)). Antiplatelet agentscurrently used in clinics can have a major bleeding side effect whichcan increase mortality and morbidity and significantly limit their use(Jackson et al, Nat. Rev. Drug. Discov. 2:775-789 (2003)). Usingantidotes is the most effective and reliable way to control drug actionand can reduce bleeding associated with current antiplatelet agent usein clinics, enhancing safety and reducing morbidity and mortality.

A technique termed “convergent” SELEX was used and a number of aptamersthat bind to VWF with high affinity were isolated. Furthermore, it wasshown that two of these clones inhibit VWF mediated platelet activationand aggregation in ex-vivo assays. Coincidentally, it has beendemonstrated that both of these functional aptamers bind to the sameregion of VWF involved in platelet aggregation using VWF SPI and SPIIIfragments. To test the characteristics of these aptamers in functionalassays, a PFA-100 instrument was utilized. PFA-100 simulates plateletfunction in whole blood under high shear stress and is particularlysensitive to VWF defects (Harrison, Blood Rev. 19:111-123 (2005)). Bothclone R9.3 and R9.14 completely inhibited platelet plug formation inPFA-100 at concentrations>40 nM (closing time>300s). Moreover, theseaptamers were tested in ristocetin, ADP, thrombin (SFLLRN peptide) andcollagen mediated platelet aggregation assays for pathway specificity.Both of these clones inhibited RIPA at >250 nM concentration but had nosignificant effect in other agonist mediated aggregation assays. Theseexperiments show that both clone R9.3 and clone R9.14 bind VWF with highaffinity and inhibit platelet aggregation through inhibition of GPIb-IX-V-VWF interaction. This interaction is especially important aroundareas of high shear stress (i.e., stenosed arteries) and is a validtarget for antiplatelet therapy.

Antidote control gives physicians added control over drug activity andprovides a safer means for antiplatelet therapy. To further improve thesafety of the lead molecule R9.14, an antidote oligonucleotide wasrationally designed using the properties inherent to nucleic acids(Rusconi et al, Nature 419:90-94 (2002), Rusconi et al, Nat. Biotechnol.22:1423-1428 (2004), Nimjee et al, Mol. Ther. 14:408-415 (2006)).Antidote oligonucleotides bind to their target aptamer throughWatson-Crick base pairing, thus changing the aptamer's conformationalshape and inhibiting binding to its target, therefore reversing itsactivity. Six different antidote oligonucleotides were designed andtheir activity tested in nitrocellulose filter binding assay. Antidoteoligonucleotide 6 (AO6) was the most effective in inhibiting aptamerbinding to VWF, completely reducing it to nonspecific, backgroundlevels. To test the effect of antidote AO6 on clone R9.14, the pair wastested in PFA-100. AO6 completely reverses the antiplatelet effect ofR9.14 in less than 2 minutes and is effective for at least 4 hours. Thisaptamer-antidote pair can potentially give physicians a rapid, effectiveand continual way to regulate antiplatelet therapy.

EXAMPLE 4

Experimental Details

Synthesis of Aptamer Truncates and Antidote Oligonucleotides

The antidote oligonucleotides and primers used in the truncation of theaptamer were synthesized and purified by IDT Inc (Coralville, Ind.).Software predicting RNA secondary structure (Mfold by M. Zuker) was usedto aid in the design of truncates. Briefly, primers were designed tomake progressively shorter DNA templates for aptamer molecules. T7 RNApolymerase was then used to transcribe RNA aptamers and tested each ofthese in binding assays.

Binding Assay

Dissociation constants (K_(d)) of each truncate were determined usingdouble-filter nitrocellulose filter binding assays (Pergolizzi et al,Blood 108:862-869 (2006)). All binding studies were performed in bindingbuffer F (20 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM CaCl₂, and 0.1% BSA) at37° C. Human purified VWF (with factor VIII and factor VIII free) waspurchased from Haematologic Technologies Inc. (Essex Junction, VT) andused in the double-filter nitrocellulose filter binding assay todetermine the K_(d) of individual clones. Briefly, RNA wasdephosphorylated using bacterial alkaline phosphatase (Gibco BRL,Gaithberg, Md.) and end-labeled at the 5′ end with T4 polynucleotidekinase (New England Biolabs, Beverly, Mass.) and [γ³²P] ATP (AmershamPharmacia Biotech, Piscataway, N.J.). Direct binding was performed byincubating ³²P—RNA with VWF in physiological buffer+1 mg/ml BSA at 37°C. for 5 min. The fraction of the nucleic acid-protein complex whichbound to the nitrocellulose membrane was quantified with a phosphoimager(Molecular Dynamics, Sunnyvale, Calif.).

Non-specific binding of the radiolabeled nucleic acid was subtracted outof the binding such that only specific binding remained (Wong andLohman, Proc. Natl. Acad. Sci. USA 90:5428-5432 (1993)).

Platelet Function Analysis

The Platelet Function Analyzer, PFA-100 (Dade Behring, Deerfield, Ill.),is a whole blood assay that measures platelet function in terms of clotformation time (Harrison, P., Blood Reviews 19:111-123 (2005)). It ishighly sensitive to VWF levels. Briefly, whole blood (840 μl) was mixedwith VWF aptamer in phosphate-buffered saline with magnesium and calcium(Sigma Aldrich, St. Louis, Mo.) and incubated for 5 minutes at roomtemperature. This mixture was then added to a collagen/ADP cartridge andtested for its closing time. The cartridge contains a microscopicaperture cut into a biologically active membrane at the end of acapillary. The whole blood is drawn through the aperture and themembrane is coated with collagen and adenosine diphosphate (ADP) orcollagen and epinephrine which activate platelests. The activatedplatelet form a plug which occludes the aperture and stops blood flow.The time it takes for this to occur represents the closing time. Themaximum closing time that the PFA-100 machine records is 300 seconds.The effect of the antidote oligonucleotides on the activity of theaptamer was measured by mixing whole blood with aptamer, incubating for5 minutes followed by addition of antidote or negative control, andtesting the mixture in the PFA-100.

Murine In vivo Studies

All in vivo experiments were approved by the Duke UniversityInstitutional Animal Care and Use Committee. All experiments werecompleted with the operator and the neuropathologist blinded to thetreatment group.

Carotid Injury

Adult C57BL/6J mice (Jackson Laboratory, Bar Harbor, Me.) (18-24 g) wereintubated and the left jugular vein was cannulated. Next, the rightcommon carotid artery was exposed and a transonic flow probe (TransonicSystems Incorporated, Ithaca, N.Y.) was placed around the vessel. Theblood flow was measured for 5 minutes to achieve a stable baselinefollowed by intravenous injection of negative control or aptamerCh-9.14-T10. Carotid artery thrombosis was activated by 10% ferricchloride-soaked Whatmann paper as previously described (Konstantinideset al, Circulation 103:576-583 (2001)). The blood flow was then measuredfor 60 minutes. The time to occlusion was recorded. The animals werethen sacrificed. The brain and common carotid arteries were harvestedfor analysis. The brain and carotid arteries were prepared on slides andstained with hematoxylin and eosin stains (Duke University Department ofPathology, Durham, N.C.) and reviewed by a neuropathologist (TJC), whowas blinded to the treatment groups.

Tail Transaction

Adult mice (18-24 g) received Ch9.14T10 or saline by intraperitoneal(IP) injection. After 5 minutes, the mice were injected with saline or10-fold molar excess of antidote oligonucleotide 1 (AO1). After 2minutes, 2 mm of the distal tail was amputated and blood was collectedfor 15 minutes in 1 mL of phosphate-buffered saline at 37° C. Blood losswas determined by measuring the absorbance of saline at 550 nm andcomparing the result to a standard curve constructed from known volumesof mouse blood as previously described (Fay et al, Blood 93:1825-1830(1999)).

Data Analysis

All data is expressed as mean±standard deviation. All data was inputtedinto Graphpad Prism (Graphpad Software, La Jolla, Calif.). Allstatistical analysis was performed using Graphpad Prism or GraphpadInstat (Graphpad Software, La Jolla, Calif.).

Results

The VWF Aptamer Inhibits Thrombosis In Vivo

Previously described aptamer termed 9.14 (Table 4) binds and inhibitshuman VWF binding to human GP Ib-IX-V and prevents human plateletaggregation in vitro (Oney et al, Oligonucleotides 17:265-274 (2007)).To evaluate the ability of this aptamer to inhibit platelet function inanimals, an attempt was first made to truncate and modify aptamer 9.14to facilitate large scale synthesis of the oligonucleotide. Truncatedversions of the aptamer were created and tested based on progressivedeletion of nucleotides from the 3′ end of the molecule (Table 4). Itwas determined that aptamer 9.14 could be truncated from 80 nucleotidesto 60 nucleotides without significantly reducing its ability to bind VWF(dissociation constant (K_(d)) of 44 nM compared to 12 nM for the fulllength aptamer) (Oney et al, Oligonucleotides 17:265-274 (2007)) (FIG.13A). Moreover, this truncated aptamer termed 9.14-T10 also retained itsability to inhibit platelet function as measured in a Platelet FunctionAnalyzer (PFA-100) assay (FIG. 13B). Truncate 9.14-T10 also tolerated acholesterol modification to the 5′-end of the aptamer (termedCh-9.14-T10) to increase its circulating half-life in vivo (Rusconi etal, Nature Biotechnology 22:1423-1428 (2004)) without altering itsability to completely inhibit platelet aggregation (FIG. 13B).

TABLE 4 Sequences and binding properties of VWF aptamer truncates LengthBmax Aptamer (nt) Binding (%) Sequence 9.14 80 70GGGAGGACGATGCGGTGGACGAACTGCCCTCAGCTACTTTCATGTTGCTGACGCACAGACGACTCGCTGAGGATCCGAGA 9.14 T1 77 Similar 72GGGAGGACGATGCGGTGGACGAACTGCCCTCAGCTACTTTCATGTTGCTGACGCACAGACGACTCGCTGAGGATCCG 9.14 T2 72 Decreased 50GGGAGGACGATGCGGTGGACGAACTGCCCTCAGCTACTTTCATGTTGCTGACGCACAGACGACTCGCTGAGG 9.14 T3 69 Decreased 50GGGAGGACGATGCGGTGGACGAACTGCCCTCAGCTACTTTCATGTTGCTGACG CACAGACGACTCGCTG9.14 T4 51 No transcriptionGGGAGGCCTCAGCTACTTTCATGTTGCTGACGCACAGACGACTCGCTGAGG 9.14 T5 66 Decreased28 GGGAGGACGATGCGGTGGACGAACTGCCCTCAGCTACTTTCATGTTGcrGA CGCACAGACGACTCG9.14 T6 63 Decreased 36GGGAGGACGATGCGGTGGACGAACTGCCCTCAGCTACTTTCATGTTGCTGACGCACAGACAGC 9.14 T759 Decreased 22GGGAGGACGATGCGGTGGACGAACTGCCCTCAGCTACTTTCATGTTGCTGACGCACAGA 9.14 T8 66Similar 67GGGAGGATGCGGTGGACGAACTGCCCTCAGCTACTTTCATGTTGCTGACGCACAGACGACTCGCTG9.14 T9 63 No transcriptionGGGAGGCGGTGGACGAACTGCCCTCAGCTACTTTCATGTTGCTGACGCACAGACGACTCGCTG 9.14 T1060 Similar 61GGGAGGTGGACGAACTGCCCTCAGCTACTTTCATGTTGCTGACGCACAGACGACTCGCTG 9.14 T11 57Decreased 58 GGGAGGADGAACTGCCCTCA.GCTACTTTCATGTTGCTGACGCACAGACGACTCGCiTG9.14 T12 54 Decreased 45GGGAGGAACTGCCCTCAGCTACTTTCATGTTGCTGACGCACAGACGACTCGCTG 9.14 T13 51No transcription GGGAGGTGCCCTCAGCTACTTTCATGTTGCTGACGCACAGACGACTCGCTG9.14 T14 28 No transcription GGGAGGTCAGCTACTTTCATGTTGCTGA 9.14 T15 57No transcriptionGGGAGGTGGACGAACTGCCCTCAGCTACCATGTTGCTGACGCACAGACGACTCGCTG 9.14 T16 54No transcription GGGAGGTGGACGAACTGCCCTCAGCTACGTTGCTGACGCACAGACGACTCGCTG9.14 T17 40 No transcription GGGAGGTGGACGAACTGCCCTACGCACAGACGACTCGCTG9.14 T18 57 Similar 79GGGAGGTGGACGAACTGCCCTCTACTTTCATGTTGCTGACGCACAGACGACTCGCTG 9.14 T19 54No transcription GGGAGGTGGACGAACTGCCCTCTTTCATGTTGCTGACGCACAGACGACTCGCTG9.14 T20 54 No transcriptionGGGAGGTGGACGAACTGCCCTCTACTTTCATGTTGACGCACAGACGACTCGCTG Abbreviations:nt, nucleotide; VWF, von Willebrand factor.

To determine if the aptamer could inhibit platelet function in vivo, anevaluation was made of its ability to limit thrombosis in a ferricchloride-induced damage model of the common carotid artery in mice.After intubation, cannulation of the left jugular vein and placement ofa flow probe around the right common carotid artery, each animalreceived an intravenous bolus injection of aptamer Ch-VWF 9.14 T10 (3mg/kg, n=11) or phosphate-buffered saline (n=11). Next, Watmann paper (1mm²) soaked in 10% ferric chloride (370 mM) was placed on the carotidartery proximal to the flow probe and left on for 5 minutes to induceendothelial damage before being removed (Westrick et al, ArteriosclerThromb Vasc Biol 27:2079-2093 (2007)). The average time to thrombosis ofthe common carotid artery in the negative control group wasapproximately 10 minutes. By contrast the carotid arteries of allaptamer Ch-9.14 T10-treated mice remained patent until the end of theexperiment (60 minutes) (p<0.0001 compared to the negative controlgroup) (FIG. 14A). Moreover, no significant change in blood flow wasobserved in aptamer treated animals from the beginning of the experimentand for the entire 60 minutes of the experiment when the procedure waselectively terminated (FIG. 14A).

Histological analysis of the carotid arteries of all the mice confirmedthat vessels in the aptamer Ch-9.14-T10 treated animals were patent anddevoid of thrombi (FIG. 14B). This observation was in stark contrast toall PBS-treated control mice, which had thrombi that had completelyoccluded their arteries (p<0.0001 of the Ch-9.14-T10-treated compared toPBS-treated controls) (FIG. 14C).

The VWF Aptamer Increases Bleeding from Surgically Challenged Animals

Once it was determined that aptamer Ch-9.14-T10 was a potentantithrombotic agent in vivo, its potential safety profile wasevaluated. Therefore, animals that had received the aptamer weresurgically challenged to determine the degree of bleeding. A murinetail-transection bleeding model was employed in which aptamerCh-9.14-T10 was administered and 5 minutes later, the animal's tailswere transected and the volume of blood lost over the next 15 minuteswas measured. As anticipated from the bleeding diathesis described inVWF-knockout mice (Denis et al, Proc. Natl. Acad. Sci. USA 95:9524-9529(1988), Pergolizzi et al, Blood 108:862-869 (2006)), mice treated withvarying doses of aptamer Ch-9.14-T10 (10 mg/kg, 5 mg/kg, 3 mg/kg and 1mg/kg, n=5 for each dose) exhibited significantly enhanced bleeding ascompared with control animals (FIG. 15) (p<0.0001 comparingaptamer-treated mice at each dose to control animals). Moreover, thiseffect was dose-dependent and most of the aptamer-treated animals didnot stop bleeding for the duration of the experiment, whereas all of thephosphate-buffered saline-treated animals formed a platelet plug at thetail transaction site and stopped bleeding within 15 minutes. Theseresults demonstrate that, as expected for a potent platelet inhibitor,aptamer Ch-9.14-T10 can lead to significant blood loss insurgically-challenged animals. By contrast, no evidence of bleeding wasobserved in the brains of normal, adult mice that had been treated withaptamer Ch-9.14-T10 (3 mg/kg) for 1 hour and not surgically challenged.These results taken together suggest that the aptamer does not causespontaneous bleeding, but can dramatically facilitate it at sites ofvascular/tissue injury.

Therefore, after determining that aptamer Ch-9.14-T10 can preventthrombosis but can also enhance bleeding, antidote molecules weredeveloped that could be used to rapidly reverse the activity of aptamerCh-9.14-T10 if needed.

Antidote Oligonucleotides and CDP Both Neutralize VWF Aptamer ActivityIn vitro.

Complementary antidote oligonucleotides were designed based on thesequence of aptamer Ch-9.14-T10 and base pairing rules (FIG. 16A).Initially, the antidotes were tested for their ability to reverse theaptamer's activity in a platelet function assay (PFA100) (FIG. 16B). Incomparison to so the other antidote oligonucleotides (AOs), AO1 was themost potent reversal agent and it completely reversed the activity ofaptamer Ch-9.14-T10 in 2 minutes (p=0.01 compared to other AO). AO1 wasthen tested at varying concentrations and it was found that the lowestconcentration of AO1 (5-fold molar excess of AO1 over aptamerCh-9.14-T10) completely is reversed aptamer activity in a PFA-100 assay.(FIG. 16C). These results demonstrate that VWF aptamer Ch-R9.14-T10 canbe completely reversed by AO1 in vitro.

Beta-cyclodextrin-containing polycation (CDP) is a polymer that can bindto nucleic acid aptamers and inhibit their activity (Oney et al, NatureMedicine 15:1224-1228 (2009)). The ability of CDP to reverse theactivity of Ch-9.14-T10 in the PFA100 was tested. As shown in FIG. 16D,it was observed that CDP could reverse the activity of the aptamer whenadded at a modest excess (>5-fold molar excess) over the aptamer.

After establishing that AO1 and CDP both inhibit the activity ofCh-9.14-110 in vitro, the activity of the aptamer and antidotes wastested in vivo.

Antidote Oligonucleotides and Universal Antidotes can Counteract theActivity of the VWF Aptamer in Mice and Thereby Limit Blood Loss inSurgically-Challenged Animals.

An evaluation was made of the ability of the antidote oligonucleotide,AO1, and the universal antidote, CDP, for their respective abilities toreverse aptamer Ch-R9.14-T10 activity in vivo. A murine tailtransaction-bleeding model was used. Animals received aptamerCh-R9.14-T10 (3 mg/kg) by intraperitoneal injection. Five minutes postaptamer administration, PBS, AO1, or CDP was injected into the tail veinat 10-fold molar excess over aptamer. The animals were then surgicallychallenged by tail transection and blood loss was monitored.Administration of AO1 or CDP completely reversed the enhanced bleedingprovoked by aptamer Ch-9.14-T10 within two minutes (p<0.0001 betweenanimals given or not given the antidote). Blood loss from the surgicallychallenged animals given the aptamer but not given the antidote was135±34 μl. By contrast, blood loss from animals treated with the aptamerand then given AO1 or CDP was reduced to 23±12 μl and 17±10 μl,respectively. This amount of blood loss is not significantly differentfrom animals surgically challenged but not administered the aptamerwhere blood loss was 22±16 μl (p>0.95 between no aptamer and aptamerplus AO1- and aptamer plus CDP-treated animals) (FIG. 17A).

Universal Antidotes can Counteract the Activity of MultipleAntithrombotic Aptamers In vivo while Antidote Oligonucleotides OnlyControl the Activities of Individual Aptamers

Since both an antidote oligonucleotide (AO1) and a universal antidote(CDP) were able to rapidly reverse the activity of the VWF aptamer andprevent aptamer-dependent bleeding from surgically challenged animals,an investigation was made as to how these control agents may differ. Ithas been previously shown that an anti-factor IXa (FIXa) aptamer termedCh9.3T inhibited coagulation FIXa activity in mice and caused increasedbleeding and that enhanced bleeding could be controlled byadministration of an antidote oligonucleotide termed 5-2C (Rusconi etal, Nature Biotechnology 22:1423-1428 (20004)). In this experiment, theobject was to determine if a universal antidote could simultaneouslyneutralize both an anticoagulant and an antiplatelet aptamer. Thisquestion was of particular interest because, in many clinical settings;such as percutaneous coronary intervention where both anticoagulants andantiplatelet agents are employed in combination. Therefore, theanti-FIXa aptamer Ch9.3T (10 mg/kg) and the VWF aptamer Ch-R9.14-T10 (3mg/kg) were administered to animals. Five minutes later, PBS or a10-fold molar excess of an antidote oligonucleotide (AO1 or 5-2C) or theuniversal antidote CDP was administered. Then animals were surgicallychallenged by tail transection as previously described. Animals treatedwith the anticoagulant and antiplatelet aptamer combination, surgicallychallenged and not given an antidote (PBS control group) lost largeamounts of blood (223±53 μl). As expected, this amount of blood loss wassignificantly greater than animals treated with the antiplateletaptamer, Ch-R9.14-T10 alone (135±34 μl, p=0.014) (FIGS. 17A and 17B).Administration of AO1 or 5-2C decreased blood loss to a similar level(148±88 μl and 125±79 μl, respectively (p=0.72)), but remained elevatedcompared to animals that did not receive either aptamer (22±17 μl,(p=0.03)).

Administration of the universal antidote CDP to animals that hadreceived both the anticoagulant and the antiplatelet aptamer,significantly reduced blood loss from surgically challenged animals(12±8 μl), a level markedly reduced compared to animals treated withboth aptamers and given one of the matched antidote oligonucleotides(p=0.009 and p=0.01 compared to AO1 and 5-2C treated animalsrespectively). Moreover this amount of blood loss was not significantlydifferent from mice that had not received an aptamer (p=0.27) (FIG.17B).

In summary, these experiments demonstrate that the VWF aptamer Ch-9.14T10 is a potent antiplatelet agent that can block thrombosis in vivo. Asanticipated, administration of this potent platelet inhibitor enhancesbleeding from animals that are surgically challenged. A matchedantidote-oligonucleotide as well as a universal antidote can rapidlyreverse the aptamer's antiplatelet activity and thereby limitsurgically-induced bleeding.

Aptamer Ch-9.14-T10 was able to maintain vessel patency for greater than1 hour in a murine FeCl₃ vascular injury thrombosis model andhistopathologic analysis of the damaged carotid artery showed minimalevidence of platelet accumulation. It was previously determined thatCh-9.14-T10 binds to the A1 region of VWF and inhibits its interactionwith platelet GP Ib (Oney et al, Oligonucleotides 17:265-274 (2007)),interfering with platelet adhesion to subendothelial collagen andimpeding a key signal transduction pathway which subsequently leads toplatelet aggregation through GP IIb/IIIa (Ruggeri, Z. M., CurrentOpinion in Hematology 10:142-149 (2003), Ruggeri et al, Blood 94:172-178(1999)). Thus, it is believed that this mechanism of VWF inhibition isresponsible for the potent anti-platelet effect observed in vivo.

While aptamer Ch-9.14-T10 exhibited potent in vivo antiplateletactivity, several antiplatelet agents have now been described with thisability (Cadroy et al, Blood 83:3218-3224 (1994), Jackson andSchoenwaelder, Nat. Rev. Drug Discov. 2:775-789 (2003)). However, thisis the first description of an antiplatelet agent that can beneutralized rapidly and effectively by administration of an antidotemolecule in vivo. Parenteral platelet inhibitors are used extensively byinterventional cardiologists and radiologists and are now being used byneurosurgeons to aid in the surgical management of stroke andstent-assisted coiling of aneurysms (Gandhi et al, Curr Treat OptionsCardiovasc Med 9:99-108 (2007), Nelson et al, Neurosurgery 59:S77-92;discussion 53 (2006), Bendok et al, Surg Neurol 62:304-311 (2004), NEngl J Med 339:436-443 (1998), Scarborough et al, Circulation100:437-444 (1999), Topol et al, Lancet 353:227-231 (1999)). Bothinterventionalists and surgeons across vascular specialties areenthusiastic to use these types of drugs more extensively but arereluctant to do so because of concern for bleeding complications.Attenuating their pharmacodynamic activity in the event of bleedingrequires blood product transfusion or recombinant clotting proteaseadministration. Unfortunately, such procedures yield protracted andunknown quantitative inhibitory effects, are potentially prothromboticand may be ineffective.

A number of agents targeting VWF, including mAbs and aptamers, have beenshown to inhibit its activity and in turn, platelet function (Cosmi, B.,Curr Opin Molec Therap 11:322-328 (2009), Diener et al, J. Thromb.Haemost 7:1155-1162 (2009), Wu et al, Blood 99:3623-3628 (2002),Yamamoto et al, Thromb Haemost 79:202-210 (1998), Cadroy et al, Blood83:3218-3224 (1994), Gilbert et al, Circulation 116:2678-2686 (2007),Mayr et al, Transfusion 50:1079-1087 (2010), Spiel et al, Platelets20:334-340 (2009)). However, antidotes have not been described for anyof these drugs, limiting their overall clinical utility in surgicalsettings. The VWF aptamer and its antidotes, including universalantidotes such as CDP represent the first controllable anti-plateletagent and can provide clinicians with much needed options in surgicalsettings where thrombotic and hemorrhagic risk coexist.

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All documents and other information sources cited above are herebyincorporated in their entirety by reference.

What is claimed is:
 1. A nucleic acid aptamer that binds to vonWillebrand Factor (VWF) with high affinity and inhibits plateletfunction in vivo, wherein the aptamer is SEQ ID NO: 56 and includes a 3′or 5′ modification, wherein said modification confers improved in vivostability and/or delivery of the nucleic acid aptamer.
 2. The aptameraccording to claim 1 wherein said aptamer inhibits binding of VWF to GP1b-IX-V.
 3. The nucleic acid aptamer of claim 1, wherein themodification is selected from a cap, PEG or cholesterol modification. 4.The nucleic acid aptamer of claim 3, wherein said modification is a 5′cholesterol modification.
 5. A composition comprising said aptameraccording to claim 1 and a carrier.
 6. A method of inhibiting plateletaggregation in a subject comprising contacting VWF with an amount ofsaid aptamer according to claim 1 so that said inhibition is affected.